This invention relates to osteogenic devices, and more specifically to synthetic implants which induce osteogenesis in vivo. More particularly, this invention relates to biocompatible, bioresorbable, synthetic matrices which promote endochondral bone growth in vivo.
The potential utility of an osteogenic device capable of inducing endochondral bone formation in vivo has been recognized widely. It is contemplated that the availability of such a device would revolutionize orthopedic medicine, certain types of plastic surgery, and various periodontal and craniofacial reconstructive procedures.
The developmental cascade of bone differentiation in mammalian bone tissue is well documented in the art (Reddi (1981) Collagen Rel. Res. 1:209-226). Though the precise mechanisms underlying the phenotypic transformations are unclear, it has been shown that the natural endochondral bone differentiation activity of bone matrix can be dissociatively extracted and reconstituted with inactive residual collagenous matrix to restore full bone inducing activity (Sampath et al. (1981), Proc. Natl. Acad. Sci. USA 78:7599-7603).
Mammalian bone tissue is known to contain one or more active factor(s) which are proteinaceous materials capable of inducing the developmental cascade of cellular events resulting in endochondral bone formation. This active factor has variously been referred to in the literature as bone morphogenetic or morphogenic protein, bone inductive protein, osteogenic protein, osteogenin, or osteoinductive protein. Recently, the protein factors referred to as osteogenic protein (OP) responsible for inducing osteogenesis have been purified, expressed in recombinant host cells, and shown to be truly osteoinductive when appropriately sorbed onto a matrix. (U.S. patent application Ser. No. 179,406).
Studies have shown that while osteoinductive proteins are active cross species, the collagenous bone matrix heretofore required for inducing endochondral bone formation is species specific (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6594). Implants of demineralized, extracted xenogenic bone matrix and OP invariably have resulted in a strong inflammatory response that has inhibited osteogenesis, presumably due to immunogenic protein components in the bone matrix. Hence, osteoinduction requiring the use of allogenic bone matrix is a serious limitation with respect to human clinical use, as human bone is neither readily available nor cost effective.
The current state of the art of materials used in surgical procedures requiring conductive bone repair, such as the recontouring or filling in of osseous defects, is disclosed by Deatherage (J. Oral Maxillofac. Surg. (1988) 17:395-359). All of the known implant materials described (hydroxlapatite, freeze-dried bone, or autogenous bone grafts) have little or no osteoinductive properties. Clearly, the ability to induce osteogenesis is preferred over bone conduction for most procedures.
U.S. Pat. No. 4,795,467 discloses a bone repair composition comprising calcium phosphate minerals and a telopeptide, reconstituted, cross-linked fibrillocollagen. U.S. Pat. No. 4,563,350 discloses an osteogenic device comprising a bone-inducing extract and a collagenous matrix composed of approximately 90% trypsinized bovine bone matrix and 10% bovine dermal collagen. U.S. Pat. No. 4,789,663 discloses a method of effecting conductive bone repair comprising exposing the defect to fresh bone, and using xenogenic collagen from bone and/or skin, wherein the collagen is enzymatically treated to remove telopeptides, and is artificially cross-linked. EPO 309,241 discloses a device for inducing endochondral bone formation comprising an osteogenic extract, and a matrix carrier comprising 60-90% mineral component and 2-40% collagen. Deatherage et al., (Collagen Rel. Res. (1987) 7:2225-2231) purports to disclose an apparently xenogenic implantable device comprising a bovine bone matrix extract and Type 1 human skin collagen. Co-pending patent application Ser. No. 422,613, filed Oct. 17, 1989, entitled xe2x80x9cBone Collagen Matrix for Xenogenic Implants,xe2x80x9d discloses a xenogenic bone implant prepared from demineralized bovine bone and including osteogenic protein.
It is an object of this invention to provide a biocompatible, biodegradable bone matrix, implantable in a mammalian host with no significant inhibitory immunogenic response. Another object is to provide a matrix which is biocompatible, biodegradable, and which is capable of inducing osteogenesis when incorporated with osteogenic protein in mammals, including humans. Still another object is to promote conductive bone growth in mammals, including humans. Yet other objects are to provide a superior material for coating implantable prothetic devices, and to increase the cellular ingrowth into such devices. Yet another object of the invention is to provide a method for the production of such matrix material.
These and other objects and features of the invention will be apparent from the description, drawings, and claims that follow.
It has been discovered that osteogenic protein dispersed within a porous, bioresorbable matrix including collagen and glycosaminoglycan is capable of inducing osteogenesis in vivo. This knowledge has been exploited to develop novel osteogenic devices disclosed herein which induce the formation of endochondral bone in a mammalian host in a shape conforming substantially to the shape of the device. The devices may also be used as a surface coating for implantable prosthetic devices to promote cellular ingrowth.
The porous matrix of the osteogenic device includes a cross-linked polymer of collagen and glycosaminoglycan. Collagen is a major protein constituent of connective tissue in vertebrates and invertebrates. Type I collagen, Type II collagen, or mixtures thereof are preferable as the matrix material of the osteogenic device. Preferably collagen comprises about 80-95% by weight of the matrix.
Glycosaminoglycans (GAGs) are mucopolysaccharides of animal origin. They are made up of residues of hexosamines glycosidically bound and alternating in a more-or-less regular manner with either hexuronic acid or hexose moietites. GAGs preferably make up at least about 5%, and more preferably, from about 6% to about 15% by weight of the polymer. Useful GAGs include those comprising sulfate groups such as chondroitin 4-sulfate, chondroitin 6-sulfate, hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate and combinations thereof. Preferably the matrix includes chondroitin 6-sulfate.
The collagen-GAG polymer is cross-linked to control the solubility and mechanical properties of the matrix. It has been determined that cross-linking the matrix to an MC value (number average molecular weight between cross-links) of about 800 to about 60,000, and preferably to an MC of between 5,000 and 10,000, is most beneficial for the osteogenic device.
The invention is embodied as a method of growing bone by conduction including contacting a viable mammalian bone with the cross-linked collagen-GAG matrix. Bone conduction is the growth of bone from existing viable bone, and involves the migration of osteoblasts from the bone to an area immediately adjacent the bone. In one aspect of the invention the matrix material is provided as a coating on an implant placed in contact with viable bone. Useful implants are composed of an inert material such as ceramic, metal, or polymer. In another aspect of the invention, conductive bone growth is induced from a viable mammalian bone by contacting the bone with matrix material into which has been dispersed a glue in an amount sufficient to solidify the matrix when implanted in a mammal or when placed at 37xc2x0 C. A useful glue is methyl cellulose. The matrix solidifies substantially in the shape of the implanted matrix.
In an alternative embodiment, osteogenic protein is dispersed within the porous matrix. Osteogenic protein comprises a pair of subunits constituting a stable dimer under oxidizing conditions. The protein may be produced using recombinant DNA techniques or may be an extract or naturally sourced purified material. In a preferred aspect, one of the subunits of this protein has an amino acid sequence sufficiently duplicative of the amino acid sequence of OP1, disclosed herein, such that the subunit, when in combination with a second suitable subunit under oxidizing conditions, induces endochondral bone in a mammal when disposed within said matrix implanted in the mammal. OP1 is a protein subunit having the amino acid sequence set forth below.
When OP1 is dimerized to form a homodimer or a heterodimer with certain other protein sequences, it can induce endochondral bone formation.
Another aspect of this invention involves methods of producing the osteogenic device which contains osteogenic protein. The method includes providing a porous matrix comprising a polymer of collagen and GAG cross-linked to an MC value of about 800 to about 60,000; and dispersing within the matrix an osteogenic protein in an amount sufficient to induce endochondral bone formation substantially in the shape of the matrix when implanted in a host.
The dispersing step may include dispersing the osteogenic protein in a solvent such as buffered saline or acetonitrile. If insoluble collagen is to be incorporated into the matrix, it, too, may be dispersed in the solvent. In one aspect of the invention, the solvent is an acidified, aqueous solution containing about 30% to about 55% acetonitrile, and preferably about 50% acetonitrile. The dispersion step may be conducted by dehydrating a mixture including the osteogenic protein and particles of the collagen-GAG polymer. Alternatively, the dispersing step may be accomplished by lyophilizing the mixture. Lyophilization is dehydration of frozen material under vacuum.
Prior to the dispersing step, the matrix may be pre-equilibrated with the solvent in which the osteogenic protein has been dispersed. The method may further include the steps of forming the product of the dispersion step into a shape with predetermined dimensions; and implanting the formed product into a mammal. Implantation of the device results in the induction of endochondral bone having essentially the shape of the formed product.
Lastly, the invention is embodied as a method of inducing endochondral bone growth in a mammal comprising the step of implanting in the mammal, either surgically or otherwise, at a location where bone formation is desired, porous matrix material containing dispersed osteogenic protein of a nature described herein.